Figure 7.
MNT loss induced in vivo in Eμ-Myc lymphomas extends survival of transplant recipients. (A) Schematic of experimental design. Mntfl/fl Eμ-Myc/CreERT2 and Eμ-Myc/CreERT2 mice were aged until they developed tumors (∼100 days). Tumor cells (Ly5.2+) were harvested and either cultured to establish cell lines for in vitro treatment with 4-OH tamoxifen (4-OHT) (see panel B) or injected intravenously into syngeneic nonirradiated C57BL/6-Ly5.1 mice for in vivo treatment with tamoxifen (see panel C). (B) 4-OH tamoxifen-induced Mnt loss enhances apoptosis of Eμ-Myc lymphoma cells in vitro. Short-term cell lines established from 2 independent Mntfl/fl Eµ-Myc/CreERT2 lymphomas (#1129 and #1271) and a control Eµ-Myc/CreERT2 lymphoma (#1194) were treated for 24 hours with or without 0.5 μM 4-OH tamoxifen and percentage annexin-V-positive cells determined by flow cytometry. Results are from 4 (#1194, #1129) or 3 (#1271) independent experiments; mean ± SD **P ≤ .01; **P ≤ .001. Immunoblot shows MNT and ACTIN protein in cells treated with tamoxifen or vehicle. (C) Tamoxifen-induced Mnt deletion significantly extends survival of mice transplanted with Mntfl/fl Eμ-Myc/CreERT2 lymphomas. Kaplan-Meier survival curves of mice transplanted with 14 independent Mntfl/fl Eμ-Myc/CreERT2 or 9 control Eμ-Myc/CreERT2 lymphomas and subsequently treated with either tamoxifen or vehicle alone. Each lymphoma was transplanted into 6 nonirradiated C57BL/6 recipients (2-4 × 106 cells/mouse), 3 of which were treated by oral gavage with tamoxifen and 3 with vehicle alone, for 2 successive days, starting on day 5; n indicates number of independent lymphomas transplanted. Significance was determined using log-rank test. See also Supplemental Figure 6A. (D) Induced Mnt deletion reduces viability of p53 wt and p53 mutant Mntfl/fl Eμ-Myc/CreERT2 lymphoma cell lines. Cell lines established from Mntfl/fl Eµ-Myc/CreERT2 and control Mnt+/+ Eµ-Myc/CreERT2 lymphomas were incubated with 0.5 μM 4-OH tamoxifen to delete Mnt, and cell viability was determined by flow cytometry (supplemental Figure 8). Viability of 4-OHT-treated cells at 48 and 72 hours is expressed relative to that of cells incubated in parallel without 4-OHT. P53 status, determined by treatment with nutlin3a, is indicated.

MNT loss induced in vivo in Eμ-Myc lymphomas extends survival of transplant recipients. (A) Schematic of experimental design. Mntfl/fl Eμ-Myc/CreERT2 and Eμ-Myc/CreERT2 mice were aged until they developed tumors (∼100 days). Tumor cells (Ly5.2+) were harvested and either cultured to establish cell lines for in vitro treatment with 4-OH tamoxifen (4-OHT) (see panel B) or injected intravenously into syngeneic nonirradiated C57BL/6-Ly5.1 mice for in vivo treatment with tamoxifen (see panel C). (B) 4-OH tamoxifen-induced Mnt loss enhances apoptosis of Eμ-Myc lymphoma cells in vitro. Short-term cell lines established from 2 independent Mntfl/fl Eµ-Myc/CreERT2 lymphomas (#1129 and #1271) and a control Eµ-Myc/CreERT2 lymphoma (#1194) were treated for 24 hours with or without 0.5 μM 4-OH tamoxifen and percentage annexin-V-positive cells determined by flow cytometry. Results are from 4 (#1194, #1129) or 3 (#1271) independent experiments; mean ± SD **P ≤ .01; **P ≤ .001. Immunoblot shows MNT and ACTIN protein in cells treated with tamoxifen or vehicle. (C) Tamoxifen-induced Mnt deletion significantly extends survival of mice transplanted with Mntfl/fl Eμ-Myc/CreERT2 lymphomas. Kaplan-Meier survival curves of mice transplanted with 14 independent Mntfl/fl Eμ-Myc/CreERT2 or 9 control Eμ-Myc/CreERT2 lymphomas and subsequently treated with either tamoxifen or vehicle alone. Each lymphoma was transplanted into 6 nonirradiated C57BL/6 recipients (2-4 × 106 cells/mouse), 3 of which were treated by oral gavage with tamoxifen and 3 with vehicle alone, for 2 successive days, starting on day 5; n indicates number of independent lymphomas transplanted. Significance was determined using log-rank test. See also Supplemental Figure 6A. (D) Induced Mnt deletion reduces viability of p53 wt and p53 mutant Mntfl/fl Eμ-Myc/CreERT2 lymphoma cell lines. Cell lines established from Mntfl/fl Eµ-Myc/CreERT2 and control Mnt+/+ Eµ-Myc/CreERT2 lymphomas were incubated with 0.5 μM 4-OH tamoxifen to delete Mnt, and cell viability was determined by flow cytometry (supplemental Figure 8). Viability of 4-OHT-treated cells at 48 and 72 hours is expressed relative to that of cells incubated in parallel without 4-OHT. P53 status, determined by treatment with nutlin3a, is indicated.

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